Peganum harmala seed extracts have been frequently reported to possess antibacterial potential through in vitro studies, but in vivo studies have acquired less attention. The present study was therefore designed to investigate its efficacy on the course of colibacillosis and effects of long-term feeding on selected parameters of general health in chickens. Two experiments were conducted in this regard. Experiment 1 (a pilot study) was performed to determine the dose of a field strain of Escherichia coli (O1:K1) required to induce clinical symptoms in 4- and 15-d-old specific-pathogen-free chickens. A successful induction of colibacillosis, in terms of clinical signs, mortality, and pathological lesions in addition to reisolation of the pathogen was observed by inoculating 4- and 15-d-old chicks with 4.3 log(10) and 6.4 log(10) cfu of E. coli, respectively, by intraperitoneal injection. Using these doses experiment 2 (main study) consisting of a single experiment with 3 parts was performed. Parts A and B generated the information regarding efficacy of the extract against infection in 4- and 15-d-old chickens applying different treatment schemes, whereas the effects of continuous feeding of the extract were assessed in part C. Whereas no protective effect of the extract could be recorded in young chickens, significant differences (P < 0.05) with regard to BW, clinical score, gross lesion score, and total granulocyte counts were observed in 15-d-old birds. Bacterial recovery per gram of tissue and reisolation frequency were lower in treated birds. The continuous feeding of the extract for 6 wk resulted in an augmentation in relative liver weight and depletion in alkaline phosphatase, protein, albumin, and globulin. It can be concluded that the crude extract of Peganum harmala possesses limited antimicrobial activity against E. coli in vivo and long-term continuous feeding may induce undesired effects. Furthermore, the study underlines the value of in vivo experiments and the diverse picture that herbal products, in this case Peganum harmala, may deliver by testing them against specific pathogens.